1. Field of the Invention
This invention relates to biological applications of alkaloids derived from the tunicate Eudistoma sp. and related compounds, to the preparation of synthetic pyridoacridines, and particularly to the synthesis of Eilatin.
2. Background of the Related Art.
In recent years it has become apparent that the sea offers an enormous biomedical potential. The marine environment is quite distinct from the terrestrial environment and a vast number of marine natural products with novel molecular architectures were already isolated and purified from diverse marine organisms. Surprisingly, only a small fraction of these novel compounds underwent detailed pharmacological and biological evaluations and only few of these were found to possess desirable biological and physiological activities, see Scheuer, "Marine Natural Products, Chemical and Biological Perspectives" Academic Press, New York, Vols. I-V, (1978-1983); Kaul et al., Ann. Rev. Pharmacol., 26, 117-142 (1986); Scheuer, Science, 248, 173-177 (1990).
The inventors have been engaged in the last 10 years in a research endeavor that involves chemical, biochemical and cell biological studies of marine natural products derived from Red Sea organisms. This interconnected effort has already yielded what others have described as "perhaps the most stimulating compounds isolated from corals and sponges"--the latrunculins. See Kaul et al., Ann. Rev. Pharmacol., 26, 117-142 (1986). The latrunculins, isolated from the Red Sea sponge, latrunculia magnifica react specifically with the actin-based cytoskeleton and appear to be the most powerful probes currently available for the pharmacological investigation of microfilament organization and function, See: Spector et al., Science, 214, 493-495 (1983); Coue et al., FEBS Lett., 213, 316-318 (1987) and Spector et al., "Cell Motility and the Cytoskeleton", 13 127-144 (1989) Also see, U.S. Pat. No. 4,857,538 to Kashman et al. entitled New Compounds for the Study and Treatment of Microfiliment Organization in cells.
Recently, two of the co-inventors, Drs. Kashman and Rudi have isolated and purified six (6) new heterocyclic alkaloids from the purple tunicate Eudistoma sp. and have elucidated their structures, See Rudi et al., Tetrahedron Lett., 29, 6655-6656 (1988); Rudi et al., Tetrahedron Lett., 29, 3861-3862 (1988) and Rudi et al., J. Org. Chem., 54, 5331-5337 (1989). The structures of these six (6) heterocyclic alkaloids (1) segoline A; (2) segoline B; (3) isosegoline A; (4) norsegoline; (5) Debromoshermilamine; and (6) Eilatin are shown in FIG. 1. The six (6) Eudistoma alkaloids have in common a fused tetracyclic benzo-3,6-diazaphenanthroline ring system that was first identified in the sponge metabolite amphimedine, see Schmitz et al., J. Am. Chem. Soc., 105, 4835-4837 (1983) and more recently in some other polycyclic aromatic alkaloids isolated from unrelated marine organisms including sponges, tunicates (ascidians), and an anemone see for example, Cimino et al., Tetrahedron, 43, 4023-4024 (1987 ); Bloor et al., J. Am. Chem. Soc., 109, 6134-6136 (1987); Cooray et al., J. Org. Chem., 53, 4619-4620 (1988); Molinsky et al., J. Org. Chem., 53, 1340-1341 (1988); Kobayashi et al., J. Org. Chem., 53, 1800-1804 (1988); Kobayashi et al., Tetrahedron Lett., 29, 1177-1180 (1988); Charyulu et al., Tetrahedron Lett., 30, 4201-4202 (1989); Molinski et al., J. Org. Chem., 54, 4256-4259 (1989); and, Schmitz et al., J. Org. Chem., 56, 804-808 (1991); Gunawardana et al., J. Am. Chem. Soc., 105, 4835 (1983); Carroll et al., J. Or. Chem., 55, 4427 (1990); and He Hay-yh et al., J. Org. Chem., 56, 5369 (1991).
Based on chemical structure, the Eudistoma alkaloids have been classified into 4 groups:
1. Three of the compounds display a high degree of structural similarity and were designated as Segoline A, segoline B, and Isosegoline A (segol means purple in Hebrew). PA1 2. A fourth compound, lacks the imide moiety that is present in the above three compounds and was designated Norsegoline. PA1 3. Another compound contains a thiazinone moiety. It was designated Debromoshermilamine A because it was found to be closely related to Shermilamine, a compound previously purified from a different tunicate trididemnum sp. found in Pago Bay, Guam, as reported by Cooray et al., J. Org. Chem., 23, 4619-4620 (1988). PA1 4. The last compound was designated Eilatin (from tunicate collected in Eilat) is the most remarkable in having a rare, highly symmetrical heptacyclic structure. PA1 (a) 12-52 .mu.M for Segoline A, Segoline B and Isosegoline; PA1 (b) 8-40 .mu.M for Norosegoline; PA1 (c) 6-32 .mu.M for Debromoshermilamine; PA1 (d) 0.05 to 0.5 .mu.M for Eilatin; PA1 (e) 7.5-12.5 .mu.M for 4-methylpyrido[2,3,4-kl]acridine; and PA1 (f) 2-5 .mu.M for pyrido[2,3,4-kl]acridine. PA1 (a) dissolving a compound having the chemical structure: ##STR15## wherein R.sub.1 is H or CH.sub.3, in acetic acid with m-nitrosulfonic acid sodium salt at a temperature of about 65.degree.-70.degree. C.; PA1 (b) adding vinylphenylketone to the solution of step (a) at a temperature of about 65.degree.-70.degree. C.; PA1 (c) heating the reaction mixture of step (b) to a temperature of about 110.degree. C..+-.5.degree. C., or reacting in an ultrasonic bath at ambient temperature, for 1.5 hrs..+-.0.5 hrs. PA1 (d) cooling the reaction mixture of step (c) to about 5.degree. C.; PA1 (e) contacting the reaction mixture of step (d) with ammoniacal ice, thereby forming a precipitate of a compound having the chemical structure: ##STR16## and, (f) separating the compound of step (e) from the mixture. The intermediate obtained in step (e) can then be used by: PA1 (g) dissolving the intermediate obtained in step (e) in 80% sulfuric acid; PA1 (h) heating the solution of step (g) to a temperature solution in an ultrasonic bath at ambient temperature, for about 1.5.+-.0.5 hrs.; PA1 (i) cooling the reaction mixture of step (h) to about 5.degree. C.; PA1 (j) contacting the reaction mixture of step (i) with ammoniacal ice; PA1 (k) extracting the resulting amine with methylene chloride; and PA1 (l) evaporating the methylene chloride yielding the free amine compound having the chemical structure: ##STR17## PA1 (m) dissolving the amine compound in 1.5N HCl; PA1 (n) cooling the solution of step (m) to about 0.degree. C.; PA1 (o) adding NaNO.sub.2 to the solution of step (n); PA1 (p) after about 20 minutes at about 0.degree. C. adding NaN the solution of step (o); PA1 (q) after about 10 minutes adding ammonia to pH 10 thereby obtaining a crude azide compound having the chemical structure: ##STR18## (r) extracting the crude azide with a solvent; (s) evaporating the solvent thereby recovering the crude azide; and PA1 (t) purifying the crude azide to yeild the pure azide compound. Preferably, in this process the solvent is methylene chloride and the purifying step is by silica gel chromotography. In continuing the synthesis from step (t), the process includes: PA1 (u) dissolving the azide in durane under argon; PA1 (v) heating the solution (u) to 200.degree. C. for approximately 30 minutes; PA1 (w) cooling (v) to about 5.degree. C.; PA1 (x) dissolving (w) in a suitable solvent; PA1 (y) extracting 2NHCl a pyridoacridine compound having the chemical structure: ##STR19## (z) neutralizing the acid with ammonia and extracting the resulting salt with a suitable solvent; PA1 (aa) evaporating the solvent to recover the pyridoacridine ocmpound; and PA1 (ab) purifying the resulting pyridoacridine compound. Preferably, the solvent of step (r) is CH.sub.2 Cl.sub.2 --CH.sub.3 OH and purifying step (ab) is by silica gel chromatography. PA1 (a) combining a compound having the chemical structure ##STR20## and o-chlorobenzoic acid under Ullmann reaction conditions to afford a substituted dephenylamine; PA1 (b) inducing cyclization of the pyridine ring of the substituted dephenylamine in the presence of poly phosphric acid (5 eq.) and at a temperature of 125.degree. C..+-.5.degree. C., or in an ultrasonic bath at ambient temperature, for about 1.5.+-.0.5 hours; and; PA1 (c) heating the reaction mixture obtained in step (b) a temperature of about 125.degree. C..+-.5.degree. C., or in an ultrasonic bath at ambient temperature, for about 1.5.+-.1/2 hrs. in the presence of catalytic amounts of sulfuric acid (H.sub.2 SO.sub.4) to yield a compound having time chemical structure: ##STR21## PA1 (d) reducing time compound obtained in step (c) with Na(Hg) to yield 1-amino-4-methylacridine. In addition, tire synthesis calm be continued by: PA1 (e) heating said 1-amino-4-methylacridine with acetyl acetone and catalytic amounts of acid (H.sub.2 SO.sub.4) in amyl alcohol at about a temperature of about 125.degree..+-.5.degree. C., or in an ultrasonic bath at ambient temperature, for about 1.5.+-.0.5 hrs. thereby yielding 1-acetyl-2,6-dimethylpyrido-(2,3,4-kl)acridine. The 1-acetyl-2,6-dimethylpyrido (2,3,4-kl)acridine is then extracted from the reaction mixture. PA1 (g) reacting said 1-amino-4-methylacridine with cyclohexanone and catalytic amounts of acid (H.sub.2 SO.sub.4) in amylalcohol at a temperature of about 125.degree. C..+-.5.degree. C. for 1.5.+-.1/2 hrs. thereby yielding a compound having the chemical structure: ##STR22## PA1 (h) extracting the compound obtained ill step (g) from the reaction mixture. PA1 (a) combining 1,8-phenathroline -5,6-dione in aniline or P-OMe-aniline (2.5 eq.) and in HOC (10 eq.), PA1 (b) reacting the mixture under reflux conditions, or in an ultrasonic bath at ambient temperature, for about 11/2.+-.1/2 hrs. thereby yielding a compound having the chemical structure: ##STR23## (c) extracting the compound obtained in step (b) from the reaction mixture; and PA1 (d) purifying the compound. PA1 (a) combining 9,10-phenanthenequinone in aniline or P-OMe-aniline (2.5 eq.) and in HOC (10 eq.), PA1 (b) reacting the mixture under reflux conditions or in an ultrasonic bath at ambient temperature, for about 11/2.+-.1/2 hrs. thereby yielding a compound having the chemical structure: ##STR24## (c) extracting the compound obtained in step (b) from the reaction mixture; and PA1 (d) purifying the compound. PA1 Reacting a first compound having the chemical structure: ##STR28## in which R.sub.10 is a substituent which may include a phenyl, halogen, hydroxy, CO.sub.2 -Methyl, HO-Methyl, COCH.sub.3, CH.sub.3, H, NHOCH.sub.3, NO.sub.2, NH.sub.2, or N.sub.3. The first compound is reacted with a second compound in a 1:2 ratio, respectively. The second compound may include kynamurine having the chemical structure: ##STR29## or derivatives of kynamurine and combinations thereof under appropriate, mild oxidative reaction conditions.
In addition to these six (6) natural compounds, Drs. Kashman and Rudi have synthesized several derivatives of Segoline A as shown in Scheme I at FIG. 2(A), Segoline B as shown in Scheme II at FIG. 2(B); see, Rudi et al., J. Org. Chem., 54, 5331-5337 (1989); also see, Gellerman, Rudi & Kashman, Tet. Letters, 33, 5577 (1992). Others have also attempted to synthesize similar compounds See, Ali et al., J. Chem. Soc. Chem. Commun., 1453 (1992)
Chemically, the six (6) Eudistoma alkaloids appear to belong to a growing class of novel marine alkaloids that have in common a fused tetracyclic benzo-3,6-diazaphenanthroline ring system. There are scant reports concerning the biological activities of these related species indicating that many of them are cytotoxic to a variety of cancer cell lines see Cimino et al., Tetrahedron, 43, 4023 (1987); Bloor et al., J. Am. Chem. Soc., 109, 6134 (1987); Molinski et al., J. Org. Chem., 53, 1340 (1988); Kobayashi et al., J. Org. Chem., 53, 1800 (1988); Kobayashi et al., Tetrahedron Lett., 29, 1177 (1988); Charyulu et al., Tetrahedron Lett., 30, 4201 (1989); Molinski et al., J. Org. Chem., 54, 4256 (1989); Schmitz et al., J. Org. Chem., 56, 804 (1991) but the mechanism by which they exert their cytotoxic effects is completely unknown.
At present it is not even clear whether all these novel marine alkaloids should be classed together, and whether they are produced by the source organism or by symbionts, see Rudi et al., J. Org. Chem., 54, 5331-5337 (1989). The presence of six different alkaloids in the same Eudistoma sp organism is without precedent and provides a unique opportunity to shed some light on these problems.